Method for identifying modulators of CLK-1 and UbiF activity

Abstract

The present invention provides methods for screening modulators of 2-polyprenyl-3-methyl-6-methoxy-1,4-benzoquinone (DMQ) hydroxylation activity, inhibitors and activators of the activity, and methods of using such compounds. More particularly, this invention relates to modulators of and UbiF and CLK-1 enzyme activity and methods of identifying same.

1. A prokaryotic test cell which is deficient in endogenous DMQ hydroxylation activity, and which comprises a target DMQ hydroxylase from a vertebrate animal. 
     
     
       2. The prokaryotic test cell of  claim 1 , which comprises a mutation or a disruption in a gene that encodes an endogenous DMQ hydroxylase, wherein said endogenous DMQ hydroxylase comprises an amino acid sequence that shares at least 50% sequence identity with SEQ ID NO: 4. 
     
     
       3. The prokaryotic test cell of  claim 2 , wherein said test cell is a  Escherichia coli  cell and said gene that comprises the mutation comprises the nucleic acid sequence of SEQ ID NO: 1. 
     
     
       4. The prokaryotic test cell of  claim 1 , wherein the transcription and/or translation of a gene that encodes an endogenous DMQ hydroxylase is inhibited, said endogenous DMQ hydroxylase comprising an amino acid sequence that shares at least 50% sequence identity with SEQ ID NO: 4. 
     
     
       5. The prokaryotic test cell of  claim 1 , which is a  Escherichia coli  cell. 
     
     
       6. The prokaryotic test cell of  claim 1 , which comprises a polynucleotide encoding a vertebrate CLK-1 protein, said polynucleotide being operably linked to a prokaryotic expression regulatory region. 
     
     
       7. The prokaryotic test cell of  claim 1 , wherein said target DMQ hydroxylase is a vertebrate CLK-1 protein comprising an amino acid sequence that shares at least 50% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15. 
     
     
       8. The prokaryotic test cell of  claim 1 , wherein said target DMQ hydroxylase is mouse CLK-1 protein or human CLK-1 protein. 
     
     
       9. The prokaryotic test cell of  claim 1 , which is JF496-mclk-1 or JF496-hclk-1. 
     
     
       10. A kit comprising the prokaryotic test cell of  claim 1 ,  3 ,  4 ,  5 ,  3 ,  6 ,  7 ,  8 , or  9  for screening for a test compound that modulates the activity of the target DMQ hydroxylase. 
     
     
       11. A method of screening for a test compound that modulates the activity of a target DMQ hydroxylase, comprising:
 (a) contacting a prokaryotic test cell with a test compound for a time sufficient to allow said test compound to modulate DMQ hydroxylase activity in said test cell, wherein said test cell is deficient in endogenous DMQ hydroxylase activity and comprises a target DMQ hydroxylase from a vertebrate animal; and 
 (b) detecting a change in DMQ hydroxylase activity, wherein an increase or decrease in DMQ hydroxylase activity in the test cell contacted with the test compound relative to the DMQ hydroxylase activity in a test cell not contacted with the test compound, indicates that the test compound modulates the activity of said target DMQ hydroxylase. 
 
     
     
       12. The method of  claim 11 , wherein said test cell is a  Escherichia coli  cell. 
     
     
       13. The method of  claim 11 , wherein said target DMQ hydroxylase is a vertebrate CLK-1 protein comprising an amino acid sequence that shares at least 50% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15 test cell is a eukaryotic test cell. 
     
     
       14. The method of  claim 11  or  12  wherein said detecting comprises determining the levels of one or more quinones in said test cell. 
     
     
       15. The method of  claim 14 , wherein said quinones are any of DMQ 9 , DMQ 10 , Q 9 , and/or Q 10 . 
     
     
       16. A method of screening for a test compound that inhibits the activity of a target DMQ hydroxylase, comprising:
 (a) contacting prokaryotic test cells with a test compound for a time sufficient to allow the test compound to modulate DMQ hydroxylase activity in said test cells, wherein said test cells are deficient in endogenous DMQ hydroxylase activity and comprise a target DMQ hydroxylase from a vertebrate animal; and 
 (b) determining the growth rate of said test cells under selective condition, wherein a decrease in growth rate of said test cells relative to test cells under the same selective condition and not contacted with said test compound, indicates that said test compound inhibits the activity of said target DMQ hydroxylase. 
 
     
     
       17. A method of screening for a test compound that enhances the activity of a target DMQ hydroxylase, comprising:
 (a) contacting prokaryotic test cells with a test compound for a time sufficient to allow the test compound to modulate DMQ hydroxylase activity in said test cells, wherein said test cells are deficient in endogenous DMQ hydroxylase activity and comprise a target DMQ hydroxylase from a vertebrate animal; and 
 (b) determining the growth rate of said test cells under selective condition, wherein an increase in growth rate of said test cells relative to test cells under the same selective condition and not contacted with said test compound, indicates that said test compound enhances the activity of said target DMQ hydroxylase. 
 
     
     
       18. The method of  claim 16  or  17 , wherein said test cell is an  Escherichia coli  cell. 
     
     
       19. The method of  claim 16  or  17 , wherein said test cell is a  Escherichia coli  cell comprising the nucleic acid sequence of SEQ ID NO: 1. 
     
     
       20. The method of  claim 16  or  17 , wherein said target DMQ hydroxylase is a vertebrate CLK-1 protein comprising an amino acid sequence that shares at least 50% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15. 
     
     
       21. The method of  claim 16  or  17 , wherein said target DMQ hydroxylase is mouse CLK-1 protein or human CLK-1 protein. 
     
     
       22. The method of  claim 16  or  17 , wherein said test cell is a JF496-mclk-1 or JF496-hclk-1. 
     
     
       23. The method of  claim 16  or  17 , wherein said selective condition comprises culturing said test cells in a minimal medium that comprises succinate or glucose. 
     
     
       24. The method of  claim 16  or  17 , wherein said selective condition comprises culturing said test cells in the presence of hydrogen peroxide or copper sulfate. 
     
     
       25. The method of  claim 16  or  17 , further comprising providing a culture of said test cells at an optical density at 600 nm of about 0.03 prior to step (a), wherein said test cells are JF496-hclk-1 or JF496-mclk-1 and said selective condition comprises culturing in M9-LB medium comprising 0.5% glucose and 1 mM hydrogen peroxide. 
     
     
       26. The method of  claim 16  or  17 , further comprising testing said test compound for toxicity. 
     
     
       27. The method of  claim 16  or  17 , wherein said selective condition comprises culturing said test cells in a minimal medium that comprises only a single source of carbon. 
     
     
       28. The method of  claim 16  or  17 , wherein said selective condition comprises culturing said test cells in the presence of oxidative stress.